Advances in bioanalysis are increasingly driven by miniaturization and multiplexing, i.e. the ability to measure many samples simultaneously. The ability to measure more analytes from smaller sample volumes, dictated in many cases by limited sample size, has led to development of miniaturized microfluidics-based sample manipulation systems and novel methods for analysis in micro-scale systems. Examples include a wide variety of existing biological and chemical measurements, for example DNA sequencing, protein analysis, single-nucleotide polymorphism (SNP) analysis, high-speed and high-resolution separations and chromatography using lab-on-chip devices, and many others. Special emphasis has been given in the past decade on development of highly parallel, miniaturized assay systems for the large-scale study of genomics and proteomics and for high-throughput screening in discovery research. Examples include 2-dimensional fixed “biochip” microarrays and, more recently, identifiable micrometer-sized particles. Both approaches depend on specific binding or capture of analytes to complementary probes on the surface of the array or particle, for example by base-pair hybridization between nucleic acids, or hydrogen bonding, hydrophobic interactions, and other binding mechanisms between polypeptides.
Microarrays and associated support technologies emerged in the 1990's and now comprise an established industry. For example, Affymetrix's GeneChip® technology is a manufacturing process that uses photolithography, solid phase chemistry, and semiconductor fabrication techniques to build hundreds of thousands of DNA sequence probes on a two-dimensional array. This technology is described in, for example, Schena, M. et al., “Quantitative monitoring of gene expression patterns with a complementary DNA microarray”, Science 270, 467–470 (1995); Shalon, D. et al., “A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization”, Genome Res. 6, 639–645 (1996); Goldberg, M. J. and Rava, R. P., “Method of manufacturing biological chips”, U.S. Pat. No. 6,309,831 (2001) and Chee, M. et al., “Arrays of nucleic acid probes on biological chips”, U.S. Pat. No. 5,837,832 (1998). Other DNA microarray technologies have been developed, for example by Hyseq, Inc., Molecular Dynamics, Inc., Nanogen, Incyte Pharmaceuticals, Inc., and others known in the art.
Fixed arrays are also being developed for proteomics, defined herein as the systematic study of the proteins in a biological system. For example, Ciphergen Biosystems' ProteinChip® array technology is a process that includes chromatography and protein characterization based upon the surface-enhanced laser desorption/ionization (SELDI). These techniques combine laser-based molecular weight determination with the use of a chemically active protein chip array. This technology is described in, for example, Hutchens, T. W., “Use of retentate chromatography to generate difference maps”, U.S. Pat. No. 6,225,047 (2001); Hutchens, T. W., “Methods and apparatus for desorption and ionization of analytes”, U.S. Pat. No. 5,719,060 (1998); and Weinberger, S. R., et al., “Current achievements using ProteinChip® Array technology”, Curr. Opin. Chem. Biol., 6(1), 86–91 (2002). A number of other protein array technologies have also been developed for example by Agilent Technologies, Inc., Zyomyx, Inc., Cambridge Antibody Technology, and others known in the art.
Independent of the type of analyte being measured, microarrays base the ability to determine the identity of each probe by its fixed position within the array. Typically, each probe is synthesized or spotted at known coordinates within a grid on the surface of a substrate or chip. These “biochip” systems can carry out large numbers of analyses simultaneously, but they suffer from known disadvantages. For example, microarrays have inherently inefficient binding kinetics. Because of poor mixing at the surface and the dimensions of a typical biochip, an analyte must cover relatively large diffusion distances to bind to its complementary probe. This results in slow diffusion of analytes to and on the surface, incomplete binding reactions, and substantial lengthening of the protocol. Additionally, the application and measurement of samples on microarrays is inherently a batch process, not particularly well suited for automation. Some types of microarrays can be expensive and difficult to customize quickly as the needs of an experiment or assay might require. Variability across a microarray can lead to degraded reproducibility and precision, requiring in some cases substantial redundancy in the measurements. Sensitivity and dynamic range are frequently reported to be problematic in the analysis of microarray data. Also, with fixed microarrays it is not typically possible to recover, sort, post-process, or perform subsequent measurements on the analyte. Finally, in most microarray systems, a reporter group such as a fluorophore is required to detect binding of an analyte. Although some advances have been reported (see for example Kayyem, J. F., “Cycling probe technology using electron transfer detection”, U.S. Pat. No. 6,063,573 (2000); Nelson, B. P., et al., “Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays”, Anal. Chem. 73, 1–7 (2001)), the use of external reporter groups requires washing the array after exposure to the sample in order to remove interfering signals from unbound reporters. The resulting measurement in most microarray systems is thus an end-point measurement; i.e. the microarray is not capable of real-time, continuous measurement of binding between analyte and probe.
The use of microparticles circumvent many of the deficiencies of 2-dimensional microarrays, primarily because the assays can be done in a small, well-mixed volume in which binding kinetics are more favorable. When the sample is well mixed, there is no location-dependent variability as is the case with fixed arrays. Further, small particle size and small sample volumes increase the local concentration of probes and reduce the diffusion length an analyte must travel in order to bind to a probe, thus increasing the speed and degree of completion of binding reactions. Microparticles can be manipulated more readily by automated sample handling systems, thus the potential for customization and automation is favorable compared to two-dimensional biochip formats.
Flexibility is a further key advantage in particle-based assays. Since individual particles can be tracked and manipulated, it is in principle possible to isolate the analyte or carry out additional measurements on a subset of the particles. Creation of custom assays is made simpler by the ability to quickly select a subset of particles for a specific purpose out of a larger master library.
There remains, however, a need for innovation in the detection of binding. As mentioned previously, although non-labeled binding detection has been reported for fixed array systems, the continuing use of external reporter groups in particle-based assays remains a key disadvantage since the associated drawbacks are the same as for fixed arrays. As will be disclosed in detail below, a key object of the present invention is to eliminate the requirement for reporter groups in particle-based assays, thus simplifying the protocol, reducing time and cost, and enabling the measurement of binding in real time.
In a typical particle-based assay system, each microparticle carries many copies of a unique probe on its surface. Because the microparticles can be in free suspension in some applications, it is not possible in those cases to link the identity of the probe to a fixed position, as done in fixed arrays. Rather, identity of the probe must be uniquely linked to the identity of the particle, thus requiring each particle to have a unique identifying label or marker. In the literature, particle identification is typically accomplished by incorporation of colored or fluorescent molecules, barcodes, or nanoparticles with distinctive fluorescent signatures. Thus, the number of particle-based assays that can be performed in parallel is limited by the number of distinguishable combinations provided by the specific labels employed, e.g. the number of fluorophores, and possibly their relative abundance.
An example of fluorescence-based particle identification is Luminex Corporation's FlowMetrix® system and Laboratory Multi-Analyte Profiling (LabMAP®) technology. This system allows up to about 100 to 1000 analytes to be measured sequentially by flow cytometry. This technology incorporates microspheres that are internally labeled with two or more distinct fluorescent dyes. The microspheres are further coded with varying combinations of intensities of the fluorophores. The process also includes a third different fluorophore integrated to a reporter molecule for quantification of reactions on the surface of the encoded microspheres. The fabrication of the encoded microspheres and the system is described in, for example, Chandler, V. S., et al., “Multiplexed analysis of clinical specimens apparatus and methods”, U.S. Pat. No. 5,981,180 (1999). Due to the relatively wide emission spectra of many fluorophores, a moderate number of patterns can be uniquely distinguished with this class of labels, typically less than 1000.
The use of nanoparticles with relatively narrow fluorescent emission spectra (described for example by Chandler, M. B., et al., “Microparticles attached to nanoparticles labeled with fluorescent dye”, U.S. Pat. No. 6,268,222 (2001); Xu, H. et. al., “Multiplexed SNP genotyping using the Qbead™ system”; a quantum dot-encoded microsphere-based assay, Nucleic Acids Research 31(8), e43 (2003)). Encoding particles with other optical markers, e.g. electrochemically deposited codes (described for example by Nicewarner-Pena, S. R. et al., “Submicrometer metallic barcodes”, Science 294, 137–141 (2001); and Walton, I. D. et al., “Particles for multiplexed analysis in solution: detection and identification of striped metallic particles using optical microscopy”, Anal. Chem. 74, 2240–2247 (2002)) can improve the multiplicity of the assay while retaining the operational advantages of particle-based assays.
The present invention relies on the interaction between light and particles possessing defined physical and optical properties. More specifically, resonant light scattering from spherical particles (interchangeably referred to herein as resonant Mie scattering because the interaction of light with the particles used in the present invention is described by Mie theory) is used to determine either or both the particle identity and the degree of binding of target species the particle surface. Theories of interactions between light and particles are provided in many references, for example Bohren, C. F., and Huffman, D. R., Absorption and Scattering of Light By Small Particles, John Wiley and Sons (1983); Kerker, M., The Scattering of Light and Other Electromagnetic Radiation, Academic Press (1969). More specifically pertaining to the present invention are treatments of resonance structures in the resonant light scattering spectrum, see for example Chylek, P. et al., “Narrow resonance structure in the Mie scattering characteristics”, Appl. Optics 17, 3019–3021 (1978); Conwell, P. R. et al., “Resonant spectra of dielectric spheres”, J. Opt. Soc. America A 1, 62–67 (1984); Probert-Jones, J. R., “Resonance component of backscattering by large dielectric spheres”, J. Opt. Soc. America A 1, 822–830 (1984); Hill, S. C. and Benner, R. E., “Morphology-dependent resonances associated with stimulated processes in microspheres”, J. Opt. Soc. America B 3, 1509–1514; Lettieri, T. R., and Marx, E., “Resonance light scattering from a liquid suspension of microspheres”, Appl. Optics 25(23), 4325–4331 (1986); Chylek, P. and Zhan, J., “Interference structure of the Mie extinction cross section”, J. Opt. Soc. America A 6, 1846–1851 (1989); and Hill, S. C. et al., “Structural resonances observed in the fluorescence emission from small spheres on substrates”, Appl. Optics 23, 1680 (1984). The development of computational methods and computer algorithms for deriving structure and property information from scattered light is described in, for example, Conwell, P. R. et al., “Efficient automated algorithm for the sizing of dielectric microspheres using the resonance spectrum”, J. Opt. Soc. America A 1, 1181–1186 (1984); Lam, C. C. et al., “Explicit asymptotic formulas for the positions, widths, and strengths of resonances in Mie scattering”, J. Opt Soc. America B 9, 1585–1592 (1992); Chylek, P., “Resonance structure of Mie scattering: distance between resonances”, J. Opt. Soc. America A 7, 1609–1613 (1990); and Guimaraes, L. G., and Nussenzveig, H. M., “Uniform approximation to Mie resonances”, J. Modern Optics 41, 625–647 (1994). Of further specific relevance to the microparticles of this invention are treatments of layered spheres, for example Kaiser, T. and Schweiger, G., “Stable algorithm for the computation of Mie coefficients for scattered and transmitted fields of a coated sphere”, Computers in Physics 7, 682–686 (1993); and Hightower, R. L. and Richardson, C. B., “Resonant Mie scattering from a layered sphere”, Appl. Optics 27, 4850–4855 (1988).
In practical applications, resonant light scattering has been used for particle size and refractive index measurements, studies of atmospheric aerosols, interstellar particles, and other measurements; for example see the two articles cited above for Conwell (1984) and Lettieri (1986); also see Ray, A. K. and Nandakumar, R., “Simultaneous determination of size and wavelength-dependent refractive indices of thin-layered droplets from optical resonances”, Appl. Optics 34, 7759–7770 (1995); Huckaby, J. L., et al., “Determination of size, refractive index, and dispersion of single droplets from wavelength-dependent scattering spectra”, Appl. Optics 33, 7112–7125 (1994); Hill, S. C., et al., “Sizing dielectric spheres and cylinders by aligning measured and computed resonance locations: algorithm for multiple orders”, Appl. Optics 24, 2380–2390 (1985); Chylek, P. V. et al., “Simultaneous determination of refractive index and size of spherical dielectric particles from light scattering data”, Appl. Optics 22, 2303–2307 (1983), and the references therein. In these references it is shown that accurate detection of fine resonance features in the scattered light spectrum requires detection methods with high spectral resolution. Typical experimental relative error in measuring the wavelength position of peaks in the earlier reports, e.g. Chylek et al., (1983) is about 1 part in 105. More recently, for example in Huckaby (1994), the relative precision of peak determination is between about 1 part in 2×106 and 1 part in 2×107.
Whitten et al., in “Morphological resonances for multicomponent immunoassays”, Appl. Optics 34, 3203–3207 (1995), describe a technique for distinguishing among antibody-coated microspheres based on their sizes as measured by resonance features in their fluorescence spectrum. The technique reported in that article was capable of distinguishing among a low number of subpopulations of particles (two in the example reported), each subpopulation having a nominal mean diameter, with relatively large differences between the two subpopulations in mean diameter (6.5 and 10 micrometers). In essence, particle diameter was used as an identifying label, the diameter being measured by fitting the observed resonance pattern with theoretical calculations. As disclosed in that article, the diameter “label” could only distinguish between two subpopulations differing substantially in mean diameter. No extension of this approach to identifying large, diverse populations of very similar microparticles is taught in that disclosure. Moreover, the methods and system of the present invention also differ substantially from the method and system taught in the Whitten et. al. disclosure. That disclosure teaches a fluorescence detection method in which the incident light is fixed in wavelength and the optical resonances occur in the scanned fluorescence spectrum. In contrast, a preferred embodiment of the present invention does not rely on fluorescence, uses a scanned incident wavelength, and detects a scattered light spectrum through means differing substantially from what is disclosed by Whitten et al.
Vollmer et al. in “Protein detection by optical shift of a resonant microcavity”. Appl. Phys. Lett. 80, 4057–4059 (2002) describe the detection of protein binding to a dielectric microparticle based upon a shift in optical resonances in the particle. The resonances were excited by evanescent coupling to an eroded optical fiber and were detected as dips in the light intensity transmitted through the fiber. The detection method described in that disclosure is not based on light scattering measurements. Moreover, the possibility of using the optical resonances for particle identification is not taught by Vollmer et al.
Hightower, R. L. and Richardson, C. B, supra, used theoretical modeling to compute the resonant response of large, layered spheres to an incident linearly polarized plane wave. Based on the results of these computations, they suggest that the sharp and unique features of the scattered light spectrum may be used in studies of immiscible fluids, adsorbed layers, coatings, and vesicles. However, use of resonant light scattering spectra for identification of microparticles or for detection in biological and chemical assays is not taught in that disclosure.
Serpenguzel et al. in “Excitation of resonances of microspheres on an optical fiber”, Optics Lett 20, 654–656 (1995) describe the measurement of resonant light scattering of solid microspheres that are excited using evanescent coupling to an optical fiber. The authors of that disclosure postulate that the measurement of these light scattering resonances may be used for performing extremely sensitive adsorption and reaction measurements between species bonded to the microsphere surface and reagents in the surrounding solution. However, how such measurements could be made is not taught in that disclosure. Moreover, the possibility of using the optical resonances for particle identification is not taught by Serpenguzel et al.
Arnold et al. in U.S. patent application Publication No. 2003/0174923 describe a method and system for detecting a substance based on a resonance shift of photons orbiting within a microshere of a sensor. The microsphere is coupled with at least one optical fiber such that the microsphere is excited by evanescent coupling to the fiber. The resonances are detected as dips in the light intensity transmitted through the optical fiber. The detection method described in that disclosure is not based on light scattering measurements. That disclosure also teaches that a plurality of microspheres may be used for multianalyte detection. However, in that disclosed method, the microspheres are kept in a fixed position attached to the optical fiber. The possibility of using optical resonances as signatures for particle identification for use in tracking the particles, and therefore the attached probes, is neither taught nor suggested by Arnold et al.
In contrast to the reports in the literature, the basis for identification of microparticles in the present invention is to effectively use the very rich information content inherent in the resonant light scattering pattern. As will be disclosed in more detail later in this application, a resonant light scattering pattern may be characterized by a diverse set of variables including but not limited to peak location, peak width, peak order, periods between peaks of different orders, and polarization-dependent spectral properties. Distinguishable resonant light scattering patterns may be realized among members of a large set of similar microparticles by varying, from particle to particle, one or more of the main parameters affecting the scattering pattern, namely the structure, composition, and dimensions of the particle. Thus, according to the present invention a very rich and diverse set of scattering patterns among members of a large population of similar microparticles is created, providing a means to distinguish and identify individual microparticles.
The principal drawbacks to existing labeling techniques include limited multiplicity, i.e., limited combinations of unique identifying features, difficulties in preparing the encoded particles, speed and accuracy of decoding, and, in some cases, cost. There is a need therefore, for a method for parallel, simultaneous, particle-based measurement of binding kinetics for moderate to large numbers of analytes. Specifically, a method is needed that exhibits one or more of the following attributes: (1) the ability to measure binding without need of external reporter moieties; (2) the ability to determine quantitatively the binding of a target analyte in real time; (3) the ability to optionally amplify the binding signal; and (4) the ability to identify and optionally track individual microparticles. Each of these represents a distinct improvement over the current practice, and taken together, provide not only improved speed, accuracy, and cost reduction for existing assays, but also enable new applications not previously possible.
The present invention solves the stated problem by providing reliable, easily manufacturable, and cost-effective methods of particle identification and binding detection, capable of high multiplicity and superior in performance to current practice. In the present invention, the microparticles are identified by a novel application of high-resolution light scattering, employing specific features in the scattering spectrum as unique identifying patterns or optical signatures. Specifically, the present invention employs resonant light scattering, also known as resonant Mie scattering, as the analytical method for both determining a particle's identity and also for determining the presence of, and optionally the degree of binding to the surface of the particle. These methods may be used together, or separately, to afford assays that are substantially improved over the state of the art.